This project concerns the molecular mechanism of syndromic and nonsyndromic hereditary hearing impairment. The research of this past year focused on the MITF gene and its gene products. MITF is a transcription factor with a basic-helix-loop-helix-leucine zipper (bHLHZip) motif whose mutations cause Waardenburg syndrome type 2A (WS2A). We have found two novel mutations in families exhibiting WS2A, which encode truncated MITF proteins. The functional properties of these truncated MITF proteins were analyzed by two independent methods: electromobility shift assay; and luciferase-reporter assay. In both assays, the mutated MITF proteins were shown to lose their function but they did not interfere with the function of wild-type MITF protein, indicating they do not have a dominant-negative effect. A probable cause of WS2A in the two families is haploinsufficiency of MITF protein. The function of the MITF gene was determined by introducing this gene into NIH/3T3 cells. This transformed NIH/3T3 cells into cells with a melanocyte-like dendritic shape. These cells were immunostained for tyrosinase and tyrosinase-related protein 1 (TRP-1), proteins specific to melanocytes. Expression of these proteins and their mRNAs was confirmed by Western-blot and Northern-blot/RT-PCR analysis. In contrast, the introduction of the TFE3 gene, which also encodes a transcription factor with a bHLHZip motif and is highly homologous to MITF protein, into NIH/3T3 cells did not make cells dendritic or induce the expression of tyrosinase and TRP-1. It was concluded that the MITF gene differentiates melanocytes.